The cellular macromolecules responsible for the expression of the neoplastic phenotype of chemically transformed human cells have been investigated. A new polypeptide found in a chemically transformed human cell line has been identified as a mutant of beta-actin which has aspartic acid at position 244 instead of glycine. Determination of the structure and organization of this beta-actin gene confirmed that the alteration of beta-actin in the transformed cells is due to a point mutation (transition) in the structural gene of beta-actin. The synthesis of the mutant beta-actin was correlated with the expression of the transformed phenotype in both variants of the transformed line and its hybrids with normal human fibroblasts. This mutation resulted in several defects in the function of beta-actin, such as increased instability, reduced incorporation into cytoskeletal elements, and decreased ability to polymerize in vitro. These defects in the beta-actin molecule were associated with the disruption and loss of some of the structural elements of the cytoskeleton, such as the actin cable network. The results suggest that a mutation in beta-actin leads the cells to express transformation by disrupting the cytoskeletal structure and its function.